Improvement of oral health in the postgenomic era: opportunities for government/industry partnerships.
Explore the source record for details and available documents.
Health Policy↗
Biomedical subjects
E F Rossomando
Publications and source records attributed to E F Rossomando.
A role for TNF in bone resorption of deciduous molars in human beings.
In this study, tumor necrosis-alpha was sampled from the gingival crevice of human deciduous molars; this was compared with values measured from the crevice of those deciduous molars missing a permanent successor, and from the crevice of deciduous ankylosed molars. Tumor necrosis-alpha was harvested from the gingival crevice with magnetic microspheres coated with tumor necrosis-alpha antibodies. The amount of bead-bound tumor necrosis-alpha was quantified with the use of an enzyme-linked immunosorbent assay. One hundred seven sites (from 41 patients) were sampled; for each patient, the normal value was compared with either the molars missing a permanent successor or ankylosed value. The tumor necrosis-alpha levels were 1.6 times higher from the crevice of ankylosed deciduous molars when compared with normal deciduous molars and 2.6 times higher from the crevice of sites with a molar missing a permanent successor. The mean and standard error mean distribution of tumor necrosis-alpha expressed as picograms was: normal molars 91 pg (standard error mean +/- 20), ankylosed molars 150 pg (standard error mean +/- 31), and missing permanent successor 236 pg (standard error mean +/- 67). Analysis of variance showed the difference among the 3 means was close to attaining significant difference (F [2.104] = 2.7905, P =.066). Multiple comparison procedures indicated that the mean for molars missing a permanent successor and the normal groups were significantly different, P =.05. The results of this study suggest tumor necrosis-alpha values are elevated in the gingival crevice of deciduous molars with ankylosis and where the permanent tooth bud is congenitally missing.
Alveolar Process↗
Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.
The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory. Twenty sites were sampled; defensin was found in 100% of the sites and ranged in amount from 270-2000 ng/site. The large local concentrations of defensins, estimated in the mg/ml range, may have major effects on the microbiology of the gingival crevice.
Adult↗
Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers.
A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.
Animals↗
Relationships between clinical parameters, Interleukin 1B and histopathologic findings of gingival tissue in periodontitis patients.
IL-1 beta is one of the cytokines which can induce bone resorption and connective tissue destruction. Previous studies demonstrated that the amount of IL-1 beta in gingival crevicular fluid (GCF) was closely related to the severity of gingival index (GI) and probing depth (PD). To further investigate whether the amount of IL-1 beta in GCF and the tissues adjacent to the periodontal pockets can reflect the degree of inflammation and destruction of these tissues, the GCF and inflamed gingival specimens were harvested from 14 periodontal patients and IL-1 beta in these samples was measured by ELISA. Clinical parameters of the sampled teeth were also recorded. Possible relations among periodontal parameters, GCF or tissue IL-1 beta and the degrees of inflammation in tissue sections were analysed statistically. The results showed that with increasing GI and PD, there was an elevation of GCF and tissue IL-1 beta activity. GCF flow also showed a tendency to increase with a higher percentage of inflammatory infiltrate. The GCF and tissue IL-1 beta activity were significantly correlated with clinical parameters (GI, PD and GCF flow), as well as with the percentage of inflammatory infiltrate and tissue alterations. With the exception of a significantly negative correlation with the extent of inflammation, no obvious relation between the GCF or tissue concentration of IL-1 beta and the state of tissue pathology could be detected. In conclusion, measurements of IL-1 beta activity in GCF or diseased tissues based on the classification of clinical parameters can reflect the degree of inflammation within periodontal disease tissue. These observations indicate that clinical parameters can provide reliable means of evaluation of the severity of periodontal disease, and that IL-1 beta plays a pivotal role in the pathogenic mechanism of periodontal tissue destruction.
Adult↗
My secret life (in the gingival crevice).
This paper describes my attempt to develop a new technology for sampling the contents of the gingival crevice. Following a brief introduction on the potential of the gingival crevice as a prognosticator of periodontal health, the problems associated with attempting to fulfill this potential are discussed. These problems include difficulties in identifying a crevice component or property that was correlated with disease, the shortcomings of the paper strip method used for sampling from the crevice, and finally difficulties in defining periodontal disease and finding clinical parameters useful for correlation with biochemical studies. A new technology for sampling the contents of the gingival crevice is described. This technology is based on immunomagnetic capture in which antibody coated magnetic microspheres are introduced into the sulcus. Following the capture of antigens, the microspheres are removed and the amount of antigen quantified by immunochemical assays. This paper describes attempts to develop this technology into a commercial product and includes experiences with raising capital and marketing the discovery. A brief discussion of the role of the scientific community, the practicing dentist and insurance coverage to the technology transfer process is included. Finally, some suggestions for improving the technology transfer process are included.
Antibodies, Monoclonal↗
Orthodontic forces increase tumor necrosis factor alpha in the human gingival sulcus.
The production of cytokines has been associated with the biology of tooth movement in animal populations. The purpose of this study was to measure tumor necrosis factor-alpha (TNF) directly in the human gingival sulcus before and after the application of an orthodontic force. To recover TNF from the sulcus, paramagnetic beads, coated with monoclonal antibodies for TNF, were introduced into the gingival sulcus of 50 teeth undergoing orthodontic tooth movement (by two force systems) in 20 patients. Retrieval was performed by a permanent magnetic device designed to fit the periodontal sulcus. The samples were taken before force application (controls), and at a fixed time after force application. The amount of immunoabsorbed TNF was quantified with an immunochemical assay. There was a greater than twofold increase in TNF recoverable from the gingival sulcus after application of orthodontic forces (mean of 12.9 ng vs 30.5 ng). A Student's t test for paired samples demonstrated statistical significance at p < 0.01. We conclude that the quantity of paradental TNF, found in human gingival sulcus, is elevated during tooth movement. The source may be from the adjacent gingiva, but more likely the compressed periodontal ligament and the resorbing bone adjacent to the root surface.
Adolescent↗
Crevicular interleukin-1 beta in moderate and severe periodontitis patients and the effect of phase I periodontal treatment.
Interleukin-1 beta (IL-1 beta), a potent stimulator of bone resorption, has been implicated in the pathogenesis of periodontal destruction. However, the relationship between cytokines and periodontal disease has not been studied sufficiently to allow definitive conclusions. The aims of this study are to investigate crevicular IL-1 beta and the clinical status of patients with periodontitis and the effect of phase I periodontal therapy on levels of IL-1 beta. For this study, 130 gingival crevicular fluid (GCF) samples were harvested from non-inflamed (15) and diseased sites (115) in 11 patients with periodontitis. The gingival index (GI) and probing depth (PD) of each site was recorded initially and one month after treatment. The amount of IL-1 beta in the GCF was measured by enzyme-linked immunosorbent assay (ELISA) using an antibody specific for this cytokine. Before treatment, IL-1 beta was found in 12 of 15 non-inflamed gingival crevices and in 112 of 115 diseased pockets. The amount of IL-1 beta varied from 4.03 to 511.12 pg/site. The average amount of IL-1 beta from diseased sites was 3-fold greater than that from non-inflamed sites. Both total amount of IL-1 beta and the GCF volume, but not IL-1 beta concentration, were found to be correlated, positively, with GI score and PD. After therapy, 63 sites from 7 patients were re-examined, and the amount of IL-1 beta in 49 of 63 sites was found to have declined. These data suggest that the amount of crevicular IL-1 beta is closely associated with periodontal status. This relationship may be valuable in monitoring periodontal disease activity.
Adult↗
Effects of ethanol concentration and incubation period at 65 degrees C on CAT activity in mammalian cell extracts.
Explore the source record for details and available documents.
Animals↗
Capillary electrophoresis: separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus.
In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.
Electrophoresis↗
Tumor necrosis factor-alpha as a biochemical marker of pain and outcome in temporomandibular joints with internal derangements.
OBJECTIVE: Previous studies have demonstrated the presence of tumor necrosis factor-alpha (TNF) in human temporomandibular joint (TMJ) synovial fluid. The present study continues the investigation of the role of TNF in TMJs with internal derangements. MATERIALS AND METHODS: Synovial fluid was obtained from 18 TMJs in 12 patients undergoing either arthroscopy (14 joints) or arthrotomy (four joints) for internal derangements. Standardized clinical data were collected preoperatively, intraoperatively, and postoperatively. RESULTS: When pain on palpation was absent, the mean preoperative TNF level was 14 +/- 6 ng/mL. When pain on palpation was present, the mean TNF level was 42 +/- 39 ng/mL (significant difference at P = .05). When the surgical outcome was poor, the mean preoperative TNF level was 26 +/- 9 ng/mL. When the outcome was within the stated guidelines for a favorable result, the mean TNF level was 12 +/- 7 ng/mL (significant difference at P = .05). In addition, a significant reduction (P = .05) in TNF following joint lavage (preoperative, 48 ng/mL to postoperative, 7 ng/mL) was found. CONCLUSIONS: The finding of a positive correlation between preoperative pain and TNF values suggests a biochemical basis for the origin of the pain associated with internal derangements. The relationship between preoperative TNF levels and surgical outcome suggests that the prognosis for surgery may be predicted by measuring biochemical markers of joint disease.
Adolescent↗
A novel method for the detection of TNF-alpha in gingival crevicular fluid.
This paper reviews results on the cytokine TNF and its role as a mediator in the pathway leading from bacteria to tissue destruction. Also reviewed is the immunoadsorption-magnetic separation technology used for the capture of TNF from the sulcus and the cluster assay used for quantification of the bead-bound TNF. A comparison of the results obtained with this technique revealed recovery of TNF in a higher percent of sites than when using the paper strips. Furthermore, the cluster assay was found to be as sensitive as the ELISA. Finally, TNF values in crevicular fluid were compared to TNF in other body fluids. The recovery of TNF at aseptic sites suggests that TNF production might be initiated by factors other than bacteria, and therefore that, through their ability to initiate TNF production, other processes might be responsible for the tissue destruction of periodontitis.
Gingival Crevicular Fluid↗
Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.
A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.
Antibodies, Monoclonal↗
Immunomagnetic separation of tumor necrosis factor alpha. II. In situ procedure for the human gingival space.
An in situ procedure has been developed for the separation of tumor necrosis factor (TNF) alpha directly from the fluid in the human gingival space. Paramagnetic beads coated with anti-TNF monoclonal antibodies were introduced into the gingival space of the subject with a polypropylene-tipped calibrated delivery system and retrieved using a permanent magnet designed to fit into the space. After retrieval, the amount of immunoadsorbed TNF was quantified using an immunochemical assay called the "cluster assay". The results indicate that following the appropriate preparation of the site, over 95% of the beads could be recovered. With this method we found that 62% of those cavities sampled contained TNF and that the values ranged from 0.10 to 13.0 ng/ml with a mean value of 1.7 ng/ml. A comparison of these values with those obtained from the same space using other methods suggests that the immunomagnetic method was more effective in retrieval of TNF. Because the separation is performed in situ we have named the procedure "chromatobiosis".
Adult↗
Assay of sialyltransferase activity by reversed-phase ion-pair high-performance liquid chromatography.
Sialyltransferases (CMP-N-acetylneuraminic acid:glycoprotein sialyltransferases, EC 2.4.99.1) are involved in the transfer of a sialic acid moiety from CMP-N-acetylneuraminic acid (CMP-NeuAc) to an oligosaccharide side-chain of an acceptor, asialoglycoprotein (AGP), according to the following reaction: CMP-NeuAc + AGP----NeuAc-O-AGP + CMP. This enzyme occurs in elevated levels in the sera of patients with a wide variety of neoplastic diseases and its assay might be useful in monitoring treatment. Radioactive CMP-NeuAc has been used in assays and the radioactive sialylated product separated and counted by liquid scintillation spectrometry. This study shows that a simple, rapid, non-radiochemically based high-performance liquid chromatographic method developed for the analysis of CMP-sialic acid synthetase can be used for the quantitation of sialyltransferase activity by monitoring simultaneously the utilization of CMP-NeuAc and the release of CMP. We describe the application of this method to assay of commercially available sialyltransferase activity and to activities from synovial, ascites and gastric fluids.
Animals↗
Correlation between tumor necrosis factor-alpha (TNF-alpha)-induced cytoskeletal changes and human collagenase gene induction.
Tumor necrosis factor-alpha (TNF-alpha) has been shown not only to induce the biosynthesis and secretion of collagenase but also to change the organization of cytoskeletal components. In the present study we explore the correlation between the biosynthesis of collagenase (by mRNA hybridization, indirect immunofluorescence and collagenolytic activity), and cytoskeletal reorganization (by rhodamine-phalloidin staining of F-actin) induced in fibroblasts by recombinant TNF (rTNF). In the concentration range of 1-100 ng/ml, rTNF increased extracellular collagenase activity 8-fold and collagenase mRNA 4-fold. In addition, whereas the collagenase mRNA was detected as early as 24 h posttreatment, the appearance of extracellular collagenase activity required 48 h. Using phalloidin to follow the organization of the cytoskeleton we observed that rTNF disrupted the parallel array of stress fibers normally observed in the perinuclear region. In contrast to the time required to affect collagenase synthesis, the effect of rTNF on stress fiber organization occurred as early as 6 h post-treatment. Finally, while the number of cells exhibiting this change increased with increasing concentrations of rTNF, a maximum of about 30% of the cells showed this effect. Interestingly, double staining studies demonstrated that both stress fiber changes and procollagenase production occurred in the same cells. This finding, together with the observation that the cytoskeletal disorganization preceded collagenase gene induction by at least 18 h is consistent with the conclusion that the organizational status of the microfilaments may have a role as a regulator of procollagenase gene expression.
Actin Cytoskeleton↗
High-performance liquid chromatography in enzymatic analysis. Opening lecture.
During the previous two decades, high-performance liquid chromatography (HPLC) has proven to be an extremely useful technique with which to study the activity of enzymes and this paper will explore some of these uses. The success of the method can be seen not only from the increase in the number of papers utilizing this technique but also from the insights gained from its use on cellular phenomena. Given this success, it is no wonder that HPLC has become the technique of choice for many biologists seeking a more quantitative understanding of biological processes. Based on past experience, there is every reason to expect that the application of HPLC to the assaying of enzymatic activities will usher in another era of fundamental discoveries in the biological sciences. HPLC is particularly well suited to the assay of one activity in the presence of other activities obviating the need for extensive and tedious purification of biological samples. This advantage makes this technique particularly well suited to those who wish to use enzymes as markers for cellular processes, as indicators of metabolic activity and as evidence of gene function. To date, well over 100 activities have been assayed by this method. The method is particularly suited to problem-solving especially in such cases as when the presence of competing reactions prevents the recovery of the expected reaction products. Of the many applications, examples will be given on the use of HPLC for (1) monitoring the activity of an enzyme in a cell-free system, (2) monitoring the flow of metabolites through a multienzyme system and (3) the detection and study of new enzymatic activities. Some generalizations about the use of HPLC methods for the analysis of enzymatic activities will be presented.
Chromatography, High Pressure Liquid↗
Isolation and characterization of the protein phosphoamidates formed by a membrane bound adenylyl transferase reaction in Dictyostelium discoideum.
1. In this paper we report the results of studies on an adenylyl transferase (AyTase) activity from Dictyostelium discoideum. 2. Previous studies suggested that this activity catalyzed the transfer of AMP from ATP to a membrane protein to form a phosphoamidate reaction product. 3. In the present study we have isolated and characterized the product of the AyTase reaction and surprisingly found two AMP labeled products by SDS-gel-filtration HPLC. 4. The apparent molecular weights of these phosphoamidates were 13.5 and 1.5 kDa. 5. In addition, because the reaction product was rapidly degraded by a phosphoamidase, experiments were undertaken using AVAMP, a synthetic phosphoamidate, as an alternative phosphoamidase substrate, to trap the reaction products. 6. As the phosphoamidase activity could be inhibited by cAMP, cyclic formycin monophosphate, an analog of cAMP resistant to hydrolysis by cAMP phosphodiesterase, was also used to trap the products. 7. Both attempts at trapping failed. 8. A model for the AyTase reaction was developed to account for the failure to trap the products and the formation of two phosphoamidates.
Adenosine Monophosphate↗